Isotope kinetics of human skin cholesterol secretion.

Specific radioactivity (SA) time curves of plasma and skin surface cholesterol collected at several skin areas were recorded in 10 patients on formula diets after single intravenous injections of radioactive cholesterol. Earliest detectable radioactivity on skin surface was found in 4-6 days; depending on the skin site, SA's peaked in 13-75 days. SA's of free cholesterol were almost always higher than those of esterified cholesterol. The general forms of the SA time curves were in keeping with the idea that plasma cholesterol is carried to the skin surface in association with the epidermal and sebaceous cells, whereby (a) cholesterol synthesized de novo is mixed with derived from plasma and (b) appearances of plasma cholesterol on the skin surface is delayed by a time factor that corresponds to the movement of epidermal and sebaceous cells from the basal layer to the skin surface. The shorter mean transit times of plasma cholesterol on skin areas rich in sebaceous glands (22-24 days on the head) than on those poor in these glands (38 days on forearms and 72 days on feet) suggest that cholesterol passes faster through the sebaceous glands than through the epidermis, and faster through thin than thick epidermis. The fraction of skin surface cholesterol (f) that is derived from plasma cholesterol was estimated by three independent methods, and comparable results were obtained. Values of f were lower on skin areas rich in sebaceous glands (0.29-0.46 on forehead) than on areas poor in these glands (0.41-0.70 for forearms; 0.60 on feet) and lower for esterified (0.27-0.33) than for free (0.39-0.48) cholesterol. These data suggest that higher proportions of sebaceous gland and of esterified cholesterol, respectively, are synthesized de novo than epidermal and of free cholesterol. In two patients it was possible to calculate that f of total skin surface cholesterol was 0.49 and 0.37, respectively, and that the maximum amount of plasma cholesterol lost through the skin was 29 and 22 mg/day, respectively. Knowing the total daily excretion of total neutral and acidic steroids in feces in these patients, and assuming a total daily urinary steroid excretion 50 mg, we estimated that no more than 3.2% of total steroid excretion occurred via the skin.

The effects of unsaturated dietary fats on absorption, excretion, synthesis, and distribution of cholesterol in man.

Cholesterol balance studies were carried out in 11 patients with various types of hyperlipoproteinemia to determine the mechanism by which unsaturated fats lower plasma cholesterol. Unsaturated fats produced no increase in fecal endogenous neutral steroids in 10 of 11 patients and no decrease in absorption of exogenous cholesterol in 5 patients who received cholesterol in the diet. In 8 of 11 patients no changes occurred in excretion of bile acids during the period on unsaturated fat when plasma cholesterol was declining. However, in 3 of 11 patients small but significant increases in bile acid excretion were found during this transitional period; in 2 others increases also occurred after plasma cholesterol had become constant at lower levels on unsaturated fat.Since the majority of patients showed no change in cholesterol or bile acid excretions during the transitional period, we propose that when excretion changes did occur they were probably not the cause of the plasma cholesterol change. Furthermore, turnover data and specific activity curves suggested that cholesterol synthesis was not influenced by exchange of dietary fats. Thus, excluding changes in excretion and synthesis, we conclude that it is most likely that unsaturated fats cause plasma cholesterol to be redistributed into tissue pools. We have also examined the possibility that cholesterol which is redistributed into tissues could be secondarily excreted as neutral steroids or bile acids. In at least 5 of 11 patients excretion patterns were consistent with this explanation. However, we cannot rule out that excretion changes may have been due to alterations in transit time, to changes in bacterial flora, or to transitory changes in absorption or synthesis of cholesterol or bile acids. Our conclusion that unsaturated fats cause a redistribution of cholesterol between plasma and tissue pools points to the necessity in future to explore where cholesterol is stored, to what extent stored cholesterol can be mobilized, and to define the factors governing these fluxes.

Usefulness of chromic oxide as an internal standard for balance studies in formula-fed patients and for assessment of colonic function.

In 35 patients maintained solely on liquid formula diets, chromic oxide has been evaluated as an internal standard for balance studies that require stool collections. In 28 patients the excretion of chromic oxide was ideal: steady states were attained in which mean daily output was 90% (or more) of mean daily intake. In these patients corrections for fecal flow could validly be applied.In patients who excreted the marker ideally, the availability of chromic oxide balance data made possible the calculation of pool sizes and turnover rates of unexcreted intestinal content. These indexes bore little relationship to the usual clinical descriptions of bowel habits. In some patient who had daily bowel movements, pool sizes were very large and daily turnover was small, i.e., a large proportion of the colonic contents was not excreted for surprisingly long periods. It is critically important for investigators to recognize this possibility when carrying out balance studies for fecal constituents that may be altered by bacterial action within the gut lumen: for instance, in 6 patients a significant inverse correlation was found between daily fecal turnover and degradative losses of large amounts of dietary beta-sitosterol.7 of 35 patients failed to attain the ideal steady state of chromic oxide excretion. These patients would not have been singled out if an internal standard had not been used. In such patients balance studies that require analysis of fecal constituents must be interpreted with great caution, since the constituents in question may be handled in the same nonideal fashion as the internal standard.

Metabolism of beta-sitosterol in man.

The metabolism of beta-sitosterol was compared to that of cholesterol in 12 patients. Sterol balance methods were supplemented by radiosterol studies, with the following results. (a) Plasma concentrations of beta-sitosterol ranged from 0.30 to 1.02 mg/100 ml plasma in patients on intakes of beta-sitosterol typical of the American diet. Plasma levels were raised little when intakes were increased greatly, and on fixed intakes they were constant from week to week. On diets devoid of plant sterols, the plasma and feces rapidly became free of beta-sitosterol. (b) The percentage of esterified beta-sitosterol in the plasma was the same as for cholesterol. However, the rate of esterification of beta-sitosterol was slower than that for cholesterol. (c) Specific activity-time curves after simultaneous pulse labeling with beta-sitosterol-(3)H and cholesterol-(14)C conformed to two-pool models. The two exponential half-lives of beta-sitosterol were much shorter than for cholesterol, and pool sizes were much smaller. Values of turnover for beta-sitosterol obtained by the sterol balance method agreed closely with those derived by use of the two-pool model. There was no endogenous synthesis of beta-sitosterol in the patients studied; hence, daily turnover of beta-sitosterol equaled its daily absorption. Absorption of beta-sitosterol was 5% (or less) of daily intake, while cholesterol absorption ranged from 45 to 54% of intake. (d) About 20% of the absorbed beta-sitosterol was converted to cholic and chenodeoxycholic acids. The remainder was excreted in bile as free sterol; this excretion was more rapid than that of cholesterol. (e) The employment of beta-sitosterol as an internal standard to correct for losses of cholesterol in sterol balance studies is further validated by the results presented here.

Cholesterol distribution in the bulk tissues of man: variation with age.

Lacking reliable data on cholesterol concentrations in muscle, adipose tissue, skin, and connective tissues (i.e., the "bulk tissues") in "normal" man, we have completed these analyses in 21 men and 8 women who died suddenly and unexpectedly; their ages ranged from 23 to 78 yr. In 11 of these subjects aged 20-40 yr, the mean cholesterol concentrations ranged from 180-440 mg/100 g dry tissue. In contrast, in 13 subjects aged 60-80 yr the values were 23-28% higher in muscle, adipose, and skin, while in dura mater, biceps, and psoas tendons the concentrations were 130, 260, and 460% higher (respectively). Esterified cholesterol in these connective tissues was found to be the major contributor, increasing 5- to 10-fold in the older group as opposed to a less than 5-fold rise in free cholesterol. In view of the large proportion of total body weight represented by these tissues, it is clear that large amounts of cholesterol can be accumulated there over a lifetime; indeed, the dense connective tissues appear to act uniquely as a trap for cholesterol, especially in the esterified form. Whether analyses of tendinous tissues in man, accessible during life, mirror the pattern of cholesterol deposition in arterial connective tissue remains to be determined.

Regulation of cholesterol metabolism in the dog. I. Effects of complete bile diversion and of cholesterol feeding on absorption, synthesis, accumulation, and excretion rates measured during life.

Six adult pedigreed dogs were studied as long as 3 yr in order to determine the effects of cholesterol feeding and of bile diversion on absorption, synthesis, and storage of cholesterol. These measurements were based on cholesterol balance and isotope kinetic studies. In the six dogs fed a "cholesterol-free" diet with their enterohepatic circulations undisturbed, the rate of cholesterol synthesis ranged from 225 to 508 mg/day (mean 325 mg/day or 12.4 mg/kg/day). In two dogs studied subsequently on cholesterol-containing diets, absorption of cholesterol averaged 81% (range 71-94%) on a dietary intake of 0.5 g/day; on high cholesterol intakes (2.7-3.0 g/day) the rate of absorption dropped to 43-51% of daily intake, but the absolute amounts absorbed were increased. Feeding of cholesterol resulted in acceleration of bile acid formation and excretion, as well as nearly total inhibition of cholesterol synthesis. These two compensatory mechanisms were sufficient to maintain zero balance of cholesterol in the face of a high cholesterol intake. Plasma cholesterol concentrations in the two dogs increased by 37 and 44%. In two other dogs bile was completely diverted into the urinary system for nearly 2 yr. When these dogs were studied on a cholesterol-free diet, the sum of acidic steroids excreted daily in urine plus neutral steroids excreted in feces was seven times as high as before the operation. Since these massive excretory losses could not have been sustained by mobilization from tissue cholesterol pools, and since the rate of disappearance of [4-(14)C]cholesterol from the plasma after single intravenous injection was greatly increased after complete biliary diversion, total daily synthesis of cholesterol must have been greatly accelerated, for synthesis was totally unsuppressed. The increased output of fecal neutral steroids could be the result of transfer of plasma cholesterol across the gut wall or due to increased synthesis in the gut. Plasma cholesterol levels were reduced in these two dogs by 20 and 27%, and triglycerides decreased by 36 and 43%. Accumulation of cholesterol in body pools in the cholesterol-fed dogs appeared to have been prevented, according to antemortem measurements: increased absorption of dietary cholesterol was exactly balanced by suppression of cholesterol synthesis and enhanced bile acid excretion. In the bile-shunted animals, depletion of tissue stores of cholesterol could not be predicted by antemortem measurements.

Regulation of cholesterol metabolism in the dog. II. Effects of complete bile diversion and of cholesterol feeding on pool sizes of tissue cholesterol measured at autopsy.

In six adult pedigreed dogs the effects of high-cholesterol diets or bile diversion on the sizes of body cholesterol pools were studied at autopsy. Total body cholesterol was determined by measuring the cholesterol content of discrete organs and of the eviscerated carcass: neither cholesterol feeding nor bile diversion had altered total body cholesterol or the cholesterol content of individual organs and tissues. These results validated the conclusion based on sterol balance data obtained during life, that high-cholesterol feeding did not lead to substantial expansion of tissue cholesterol pools. THE TOTAL AMOUNT OF EXCHANGEABLE CHOLESTEROL IN THE ANIMALS WITH AN INTACT ENTEROHEPATIC CIRCULATION, WHEN ESTIMATED FROM ISOTOPIC DATA, WAS ESSENTIALLY THE SAME AS THAT MEASURED CHEMICALLY: this indicated that there was little or no nonexchangeable cholesterol in these dogs, except in skin and nervous tissue, regardless of the cholesterol content of the diet. This correspondence of estimates was not obtained in the bile-diverted dogs: we propose that the defect in the isotopic estimates was due to the accelerated rate of cholesterol synthesis in these animals. Gross and microscopic morphology of all organs and tissues was examined. Abnormal findings were limited to the biliary tract and the urinary collecting system of the two bile-diverted dogs: multiple bilirubinate gallstones were found, and mild pyelitis and ureteritis were present on the side of the bilio-renal shunt, but the urinary bladder was normal. Histologic evidence of moderate degree of cholangitis was found in one of the two bile-shunted dogs, but in neither dog was there evidence of impedance of bile flow.

Cholesterol metabolism in human obesity.

An experiment was undertaken to test whether in severe obesity cholesterol production rates obtained by isotope kinetic analysis (two-pool compartmental analysis) are comparable to those measured by chemical sterol balance techniques. Eight severely obese but normocholesterolemic patients were studied by the balance method, and five of these eight were studied by compartmental analysis. Cholesterol turnover was 10% higher by compartmental analysis. In the entire group of eight patients cholesterol turnover was greater than twice that found previously in nonobese patients studied under similar conditions with bile acids and neutral sterols both participating in the increase. This increment was directly related to excess body fat and to adipose cellularity, with correlation co-efficients of 0.66 and 0.72, respectively. The amount of cholesterol in the slowly turning over pool B was related to degree of adiposity, but that in plasma and in pool A did not differ from values in nonobese patients.

Effects of neomycin on absorption, synthesis, and/or flux of cholesterol in man.

The mode of action of the hypocholesteremic drug neomycin (2 g/day) was studied in four patients. All showed a significant reduction in plasma cholesterol concentrations (mean 25 percent, range 18-31 percent), and in one of three patients with hyperglyceridemia there was a decrease of plasma triglycerides of 26 percent. Cholesterol absorption was measured in three of four patients: there was a marked decrease. Sterol balance studies in four patients showed an unabating increase in fecal neutral steroid excretion (mean increase 345 mg/day, range 323-361) for 3-5 wk after plasma cholesterol levels had reached a new and lower plateau. Fecal acidic steroid excretion increased temporarily in two patients, with a sustained increase of 93 mg/day in only one. Daily stool weights increased significantly in three of four patients, though none had steatorrhea; there was a significant reduction in excretion of secondary bile acids; neutral sterol degradation rates were not affected by the drug. Slopes of plasma cholesterol-specific activity time curves did not change. These results fail to support the suggestion that neomycin acts as a bile acid precipitant. The finding of increased fecal neutral steroid excretion is consistent with decreased cholesterol absorption, but also with increased cholesterol absorption, but also with increased cholesterol synthesis (secondary to release of negative feedback control), with increased flux of cholesterol from tissues, or with a combination of all three actions.

Role of the kidneys in the metabolism of plasma mevalonate. Studies in humans and in rhesus monkeys.

Studies were carried out in humans and in rhesus monkeys to determine the role of the kidneys in the metabolism of circulating mevalonic acid (MVA). Following intravenous infusion of [14C]MVA and [3H]cholesterol, there was a rapid appearance of [14C]squalene in the kidneys that exhibited a significantly longer half-life than plasma or hepatic squalene. In man and in rhesus monkeys there was a rapid equilibration between newly synthesized cholesterol from MVA and exogenously administered cholesterol in all tissues except the kidneys, where the specific activity ratio of newly synthesized to exogenous cholesterol was significantly higher. Estimates of the quantitative metabolism of intravenously infused radiolabeled MVA in the monkey demonstrated that 23% was excreted in the urine, 67% metabolized to cholesterol (58% in nonrenal tissues and 9% in the kidneys), and 10% catabolized to CO2 and nonsteroid products. Measurements of MVA metabolism in anephric and uninephric patients demonstrate that, in the absence of renal uptake of MVA, exogenous and newly synthesized cholesterol achieve almost instantaneous equilibrium in the plasma; whereas in control subjects with normal renal function, this equilibration required at least 21 d for the two cholesterol decay curves to become parallel. These results suggest that the kidneys are solely responsible for the observed disequilibrium between newly synthesized and exogenous cholesterol; we suggest that this was due to the delayed release of newly synthesized cholesterol from the kidneys into the plasma compartment following intravenous infusion with radiolabeled MVA. The data document the importance of the kidneys in the metabolism of circulating MVA. However, calculation of the quantitative significance of this pathway in relation to whole body MVA metabolism indicates that renal metabolism of MVA accounts for approximately 0.1% of daily MVA turnover, and that alterations in this pathway due to any form of renal pathology would not result in significant changes in hepatic or whole body sterol synthesis rates. We urge caution in the use of radiolabeled MVA in long-term kinetic studies of sterol metabolism because our data show that the plasma compartment of MVA is not necessarily in isotopic equilibrium with tissue MVA.

Heterogeneity of cholesterol homeostasis in man. Response to changes in dietary fat quality and cholesterol quantity.

Studies were carried out to examine the effects of dietary fat and cholesterol on cholesterol homeostasis in man. 75 12-wk studies were carried out during intake of 35% of calories as either saturated or polyunsaturated fat, first low and then high in dietary cholesterol. Dietary fat and cholesterol intakes, plasma lipid and lipoprotein levels, cholesterol absorption and sterol synthesis in isolated blood mononuclear leukocytes were measured during each diet period. In 69% of the studies the subjects compensated for the increased cholesterol intake by decreasing cholesterol fractional absorption and/or endogenous cholesterol synthesis. When an increase in plasma cholesterol levels was observed there was a failure to suppress endogenous cholesterol synthesis. Plasma cholesterol levels were more sensitive to dietary fat quality than to cholesterol quantity. The results demonstrate that the responses to dietary cholesterol and fat are highly individualized and that most individuals have effective feedback control mechanisms.

Plasma mevalonate as a measure of cholesterol synthesis in man.

Measurement of mevalonic acid (MVA) concentrations in plasma or 24-h urine samples is shown to be useful in studies of the regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and cholesterol synthesis. Plasma MVA concentrations, measured either at 7-9 a.m. after an overnight fast, or throughout the 24-h cycle, were compared with cholesterol synthesis rates that were measured by the sterol balance method: plasma MVA concentrations were directly related to the rate of whole body cholesterol synthesis (r = 0.972; p less than 0.001; n = 18) over a tenfold range of cholesterol synthesis rates. Moreover, hourly examination of MVA concentrations throughout the day demonstrated that interventions such as fasting or cholesterol feeding cause suppression of the postmidnight diurnal rise in plasma MVA concentrations, with little change in the base-line of the rhythm. Thus, the daily rise and fall of plasma MVA appears to reflect changes in tissues and organs, such as the liver and intestine, that are known to be most sensitive to regulation by fasting or by dietary cholesterol. The hypothesis that short-term regulation of HMG-CoA reductase in tissues is quickly reflected by corresponding variations in plasma MVA was tested by using a specific inhibitor of HMG-CoA reductase, mevinolin, to block MVA synthesis. Mevinolin caused a dose-dependent lowering of plasma MVA after a single dose; and in patients who received the drug twice a day for 4 wk, it decreased 24-h urinary MVA output. Significant lowering of plasma cholesterol was achieved through administration of mevinolin at doses that only moderately limit MVA production.

Validation of a dietary record system for the estimation of daily cholesterol intake in individual outpatients.

In order to develop a reliable system for measuring daily cholesterol intake in individual outpatients, studies were undertaken to establish the shortest time period (in days) for which it is necessary to obtain daily food intake records. Three volunteers were trained in dietary record-keeping and portion-size assessment, and instructed to self-select a low-cholesterol diet for 20 days. During the study period they maintained daily dietary records and collected dummy diets. Comparisons of cholesterol intake calculated from the dietary records (mean 144 mg/day, SD +/- 13, n = 60) to the values from chemical analysis (118 +/- 28 mg/day) demonstrated that the calculated values were higher (mean 19%). More importantly, it was found that a minimum of 9 days' records of dummy diet analyses were required in order to reach an estimate of daily cholesterol intake that varied by less than 10% from the mean of the 20-days' values. In 100 outpatients trained to adhere to a moderately low-cholesterol intake and who maintained sequential dietary records for 9 days, it was found that the mean daily intake was 251 mg/day but that individual patients exhibited substantial daily variations in cholesterol intake (average coefficient of variation = 54%, range = 8.5 to 121.2%). These results demonstrate that, under conditions of training in dietary record-keeping and portion-size assessment, adherence to a low-cholesterol diet, and with collection of at least 9 days of dietary records, a reliable quantitative estimate of daily dietary cholesterol intake can be obtained in free-living outpatient populations.

Removal of low-density lipoproteins in patients by extracorporeal immunoadsorption.

A new technique called LDL-pheresis was used in patients to lower low-density lipoprotein cholesterol levels. This procedure combines continuous extracorporeal plasma separation with immunoadsorption of low-density lipoprotein on columns containing monospecific antibody to human apolipoprotein B. Six patients underwent a total of 164 procedures without significant side effects or nonspecific protein depletion. Acutely, LDL-pheresis lowered plasma cholesterol levels by removing up to 82 percent of the circulating low-density lipoprotein. Weekly LDL-pheresis combined with a portacaval shunt in a patient with homozygous familial hypercholesterolemia resulted in normalization of plasma cholesterol levels and rapid regression of skin xanthomata. Three of four patients with atherosclerotic coronary artery disease have noted improvement in their angina. LDL-pheresis appears to be a promising new technique capable of safely and efficiently lowering plasma low-density lipoprotein cholesterol levels.

The composition of a simulated American diet. Comparison of chemical analyses and estimates from food composition tables.

Chemical analyses were carried out on a food mixture that approximated the average daily per capita use of foods in the U.S. as of 1965-66. The food items selected were chosen from a list (items and quantities brought into the average household) generated by the USDA's 1965 Household Food Consumption Survey. Our analyses are compared with food table calculations of the items in the homogenate and with the Recommended Dietary Allowances (RDAs). Data are given for total and constituent energy supplies per person per day. The protein:fat:carbohydrate distribution of energy was found to be 18:44:38%. Unavailable carbohydrates totaled 19.1 gm. per person per day (6.8% of total carbohydrate); available carbohydrate fraction is itemized in terms of mono- and di-saccharides, starches, and dextrins. Fat content was 135 gm. per person per day; analyses showed that 16:0, 18:0, 18:1, and 18:2 fatty acids made up 89% of total fatty acids, with a P:S ratio of 0.4. Cholesterol content was 576 mg. per person per day; plant sterols, 228; beta-sitosterol, 165; and squalene, 28. Protein content was 115 gm. per person per day (over twice the recommended allowance); amino acid analyses showed concordance with the RDA estimated requirements of the essential amino acids. Major mineral and vitamin contents are also reported. Calculations and chemical data agreed well in many instances, the largest differences being for total protein, dietary "fiber," and certain vitamins.

Suitability of white-tailed deer as hosts for cattle fever ticks (Acari: Ixodidae).

White-tailed deer (Odocoileus virginianus L.) were evaluated as an alternate host for the cattle fever tick, Boophilus annulatus (Say). The total of adult female ticks recovered and associated reproductive parameters were compared between deer and bovine hosts. Significantly fewer ticks were recovered from deer than from cattle; this was attributed to the grooming behavior of the deer. The mean weight of ticks taken from deer (243.5 mg) was significantly less than that of ticks recovered from cattle (344.5 mg) with about 15% of the ticks from deer prematurely detached. Egg mass weight from the ticks recovered from deer (129.4 mg) was significantly less than that of ticks from cattle (172.1 mg). A significant difference was observed between the percentage hatch of the eggs of ticks from deer (63.1%) and ticks from cattle (76.7%). The index of reproduction was about 20 times greater for the ticks from the cattle than for the ticks from the deer. When the larval progeny of the ticks recovered from deer were reared on cattle, significantly fewer adult females were recovered than when larvae of ticks recovered from cattle were reared on cattle. This indicates a possible reduction in viability. All other parameters were comparable between the two groups. Thus, deer are biologically suitable hosts for B. annulatus, but significantly fewer ticks complete engorgement and those that complete engorgement have reduced fecundity.

Therapeutic and persistent efficacy of fipronil against Boophilus microplus (Acari: Ixodidae) on cattle.

Cattle infested with all parasitic life-stages of Boophilus microplus (Canestrini) were treated once with a pour-on formulation of fipronil at 0.25, 0.5, or 1.0% active ingredient (AI) to determine the therapeutic efficacy. Additionally, cattle were infested at weekly intervals for 13 consecutive weeks after treatment to determine the persistent effectiveness of the three concentrations. Results of therapeutic efficacy of fipronil showed that with each increase in concentration there was a corresponding and often significant (P < 0.05) decrease in the numbers, fecundity and fertility of engorged females. The overall control of the Index of Reproduction (IR) achieved at 0.25 and 0.5% AI was 86.2 and 94.3%, respectively, while the control of the IR at the 1.0% AI concentration was 99.7%. The persistent efficacy of fipronil, like the therapeutic efficacy, was also dose related. The 0.25% AI treatment failed to provide complete protection (100%) against larval reinfestation, although control of the IR remained high (> 95%) for the first 6 weeks after treatment. In contrast, at 0.5% AI the control of the IR remained at 100% for the first 4 weeks after treatment, and was > 90% for an additional 2 weeks (6 weeks posttreatment). The 1.0% AI concentration afforded the longest residual activity, providing virtually 100% protection against larval reinfestation for 8 weeks after treatment was applied. Based on the results, neither the 0.25 or 0.5% AI concentration completely eliminated the immature stages of the tick (nymphs and larvae) on the host at the time of treatment (therapeutic efficacy), and the residual activity (persistent efficacy) did not prevent reinfestation by larvae for long periods after treatment. While the 1.0% AI concentration provided both therapeutic and residual efficacy that would be suitable for use in the eradication program, caution is still warranted because a few engorged females detached for up to 25 days after treatment, although none reproduced after the 6th day.

Efficacy of amitraz (Taktic 12.5% EC) as a dip for the control of Boophilus microplus (Canestrini) (Acari: Ixodidae) on cattle.

Four groups of cattle infested with Boophilus microplus (Canestrini) were each dipped in a different concentration of amitraz diluted from a 12.5% EC formulation to determine the efficacy and performance of the product in an 11,400 l dipping vat. Except for the period when heifers were dipped, animals were restrained in stanchions placed individually inside 3.3 x 3.3 m2 stalls within an open-sided barn. The amitraz in the vat was stabilized with hydrated lime to maintain a pH of ca. 12. Analyses of vat samples showed that concentrations of amitraz in the vat were 7.6 to 13% lower than the targeted concentrations of 0.010, 0.015, 0.020, and 0.025% active ingredient (AI) for dilutions prepared according to instructions on the manufacturer's label. The large quantity of hydrated lime added to the vat (10 kg/1000 l) interfered with the HPLC analysis of vat samples. Therapeutic efficacy of each of the four observed concentrations (0.0088, 0.0131, 0.0174, and 0.0231% AI) of amitraz was excellent (> 99% control). However, the rapid detachment of all ticks from an animal within a few hours after treatment with amitraz, that has been frequently observed, was not pronounced in the present study. Only 47% of the B. microplus detached in the first 4 h post-treatment, and 84% detached within the first 24 h. All of the treatments, except the lowest concentration, provided protection of cattle against re-infestation by B. microplus larvae for 14 days post-treatment. Possibly as a result of the formation of a compact layer of lime and amitraz on the bottom after the vat was undisturbed for six weeks, intense agitation was required to re-suspend the active ingredient.

Control of Boophilus ticks on heifers with two pyrethroids applied as sprays.

The pyrethroid permethrin (25% cis to 75% trans) was sprayed on Hereford heifers infested with all parasitic stages of Boophilus microplus (Canestrini) at 0.05% and 0.1% active ingredient. At 0.05%, control was 66.7%; whereas at 0.1%, control was 93.3%. Replete females were collected daily for 28 days after treatment at both concentrations, indicating that at least some ticks treated as larvae or nymphs were able to reach repletion. The pyrethroid fenvalerate was sprayed on Hereford heifers infested with all parasitic stages of B microplus or B annulatus (Say) at 0.025% and 0.05% active ingredient. Fenvalerate was as effective as permethrin at a lower concentration. At 0.025%, control was 89.2% and 86.6% and at 0.05% was 92.5% and 90.1% for B microplus and B annulatus , respectively. Both these pyrethroids could provide adequate reduction in programs in which control is the aim; however, in an eradication program, both acaricides failed to provide the necessary reduction to achieve complete elimination.

Borrelia theileri: isolation from ticks (Boophilus microplus) and tick-borne transmission between splenectomized calves.

The bovine spirochete, Borrelia theileri, was detected in Giemsastained blood smears from a splenectomized calf 17 days after exposure to a laboratory colony of the tropical cattle tick, Boophilus microplus. Spirochetes were detected in the hemolymph and ovary of all engorged female ticks examined, indicating a high infection rate in this tick colony. Spirochetes were detected in a 2nd splenectomized calf 15 days after exposure to the larval offspring of ticks from the 1st calf. The only observable effect of infection in the 2 calves was a maximum rectal temperature increase to 40.2 C, which coincided with the first detectable parasitemia. The tick colony did not have any adverse effects, despite extensive multiplication of spirochetes in their tissues.